Perfusion-Based Tissue Harvesting Protocol

Tissue was harvested using our well-established two-stage perfusion protocol allowing for the harvest of both live tissue and fixed tissue (Amatrudo et al., 2012 (link)) for parallel experiments not in the present study. After sedation with ketamine hydrochloride (10 mg/kg) the monkeys were deeply anesthetized with sodium pentobarbital (to effect, 15 mg/kg, i.v), and then perfused through the ascending aorta with ice-cold Krebs–Henseleit buffer containing (in mM): 6.4 Na₂HPO₄, 1.4 Na₂PO₄, 137 NaCl, 2.7 KCl, 5 glucose, 0.3 CaCl₂, and 1 MgCl₂, pH 7.4 (Sigma-Aldrich). Fresh tissues were collected from the left hemisphere for parallel biochemical and electrophysiological studies not in the present study. Once the fresh tissue harvest was complete, the perfusate was switched to 4% paraformaldehyde in 0.1M phosphate buffer (PB, ph 7.4, at 37°C) to fix the remaining whole brain. The fixed brain sample was blocked, in situ, in the coronal plane, removed from the skull, cryoprotected in a series of glycerol solutions, and flash-frozen in −70°C isopentane (Rosene et al., 1986 (link)). The brain was cut on a freezing microtome in the coronal plane at 30 or 60 μm and stored in cryoprotectant (15% glycerol, in 0.1M PB, pH 7.4) at −80°C (Estrada et al., 2017 (link)).